There is considerable unmet need for novel antibiotics due to the rise of multi-drug resistant pathogens and the threat of engineered bioweapons. There is also an urgent need for novel antibiotics that can act against slow-growing or dormant pathogens and persisting biofilms. The long term goal of this screening campaign is to develop a novel method to identify antimicrobial prodrugs. ..more

There is considerable unmet need for novel antibiotics due to the rise of multi-drug resistant pathogens and the threat of engineered bioweapons. There is also an urgent need for novel antibiotics that can act against slow-growing or dormant pathogens and persisting biofilms. The long term goal of this screening campaign is to develop a novel method to identify antimicrobial prodrugs.

E. coli is a gram negative bacterium that has an outer membrane barrier and multidrug efflux pumps (MDRs) that can reduce the intracellular concentrations of potential antibiotics. TolC is an outer membrane porin that contributes to antimicrobial resistance because the major MDRs use it as an outward channel; consequently tolC deletion mutants are expected to be more sensitive to antimicrobials. As a strategy to increase the hit rate for compounds with antimicrobial activity, we screened the NIH Small Molecule Repository using an E. coli strain with mutations in TolC, referred to as E. coli ΔtolC. Compounds were screened at a final concentration of 10 uM in a standard bacterial growth assay using optical density as a measure of growth.

Actives were defined as compounds that showed ≥60.37% growth inhibition in the assay. A mathematical algorithm was used to determine nominally inhibitory compounds in the primary screen. Two values were calculated: (1) the average percent growth inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % growth inhibition than the cutoff parameter was declared active. Compounds were screened in single dose and there is an inherent uncertainty associated with a single determination.

Possible artifacts in this assay include, but are not limited to, compounds that absorb at OD615 or precipitate.Outcome: Actives were defined as compounds that showed ≥60.37% inhibition in the assay. Compounds that were less than 60.37% inhibitory were Inactives.Because of the inherent error in all high throughput screens, compounds that were active in this single dose screen were assigned a score of 100. All other compounds were assigned a score of 0.